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The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

ABSTRACT: Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its ΔlasR-derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain.IMPORTANCE Pseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections.

SUBMITTER: Soto-Aceves MP

PROVIDER: S-EPMC7890550 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Among various environmental stresses, the depletion of inorganic phosphate, which is an important signaling molecule that modulates virulence production, is notable. In our previous study, we demonstrated that lapA, encoding alkaline phosphatase, plays a key role in Pseudomonas aeruginosa biofilm formation in porcine skin explants, an ex vivo chronic wound model. However, the mechanism by which lapA participates in the virulence and biofilm formation of P. aeruginosa remains unclear. In the present study, the lapA deletion mutant was constructed in P. aeruginosa PAO1, and the phenotypic characterization was assessed. Our data demonstrated that phosphate depletion stress significantly enhanced elastase activity, hemolysis, rhamnolipid production, swarming and swimming motilities, and 3-oxo-C12-homoserine lactone (HSL) production in P. aeruginosa. Moreover, phosphate starvation evidently induced the virulence of P. aeruginosa in Caenorhabditis elegans (C. elegans) through fast-kill and slow-kill infections. Deletion of the lapA gene led to broad phenotypic changes, including reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, as well as increased chitinase activity and rhamnolipid production under phosphate depletion stress, which was unrelated to C4-HSL. Deletion of the lapA gene also significantly inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA deletion also reduced the virulence of PAO1 in C. elegans in fast-kill and slow-kill infection assays. Thus, the findings suggest that targeting lapA can help control biofilm formation and reduce virulence in nonhealing-infected wounds, where P. aeruginosa is a persistent problem. IMPORTANCE Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

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The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

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